β-mannanase from coffee berry borer, hypothenemus hampei, and uses thereof

ABSTRACT

The present invention relates to an isolated β-mannanase protein having an amino acid sequence which is 90% similar to the amino acid sequence of SEQ ID NO: 1, as well as isolated polynucleotides encoding the β-mannanase protein, and isolated expression systems and host cells containing the polynucleotides. The present invention also relates to a method of recombinantly producing β-mannanase protein. Also disclosed is a method of degrading mannans and polysaccharides in plant material, which involves providing plant material and contacting the plant material with the β-mannanase protein of the present invention under conditions effective to degrade mannans and polysaccharides in the plant material.

This application is a division of U.S. patent application Ser. No.11/943,015, filed Nov. 20, 2007, which claims the priority benefit ofU.S. Provisional Patent Application Ser. No. 60/866,705, filed Nov. 21,2006, which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to an isolated β-mannanase protein and usesthereof.

BACKGROUND OF THE INVENTION

Mannanases are enzymes that hydrolyze mannans and related hemicellulosicpolysaccharides, such as galactomannan and glucogalactomannan (alsotermed galactoglucomannan). These polysaccharides are characteristiccomponents of plant cell walls and, so, an important potentialcommercial use of mannanases is in the degradation of hemicellulosicmaterials from plant biomass, thus providing a means to recover solublesugars from these biopolymers. Mannan polysaccharides are also found asstorage polymers in the seeds of some plant species, such as those ofleguminous plants, and coniferous trees.

In coffee bean, galactomannans accumulate to extremely highconcentrations and represent approximately 24% of the dry weight of thebean (Bradbury et al., “Chemical Structures of Green Coffee BeanPolysaccharides,” J. Agric. Food Chem. 38:389-392 (1990)). Thesepolysaccharides consist of a linear chain of mannosyl residues that arelinked to each other via beta 1,4 glycosyl linkages, to which areattached alpha-galactosyl residue monomers. It is known thatendo-beta-mannanases (EC 3.2.1.78) hydrolyze mannan polymers during seedgermination, thus facilitating the exit of the rootlet duringgermination and releasing small oligosaccharides which are then used asa source of energy for the growth of the young plant. Indeed, in severalplants, it has been shown that endo-β-mannanase activity is mainlydetected in the endosperm of seeds undergoing germination (Bewley,“Breaking Down the Walls—A Role for Endo-β-Mannanase In Release fromSeed Dormancy?” Trends Plant Sci. 2:464-469 (1997)).

Mannanases are produced by microorganisms such as molds, yeasts, andfungi, as well as Bacillus subtilis, Aeromonas, Enterococcus,Pseudomonas, and Streptomyces. Some higher plants or animals can alsoproduce mannanases; however, no report exists in the literaturedescribing a β-mannanase from insects. Microorganisms that are typicallyused for commercial production of mannanases include Trichoderma orAspergillus spp.

In industrial processes, during the treatment of coffee, mannans andtheir derivatives constitute a considerable portion of the insolublesediments. In addition, during the first extraction step in coffeeproduction only approximately 50% of the mannans are soluble and thesepolymers are therefore responsible for the majority of the secondaryprecipitations which occur during the subsequent steps. European PatentNo. 0676145A demonstrated that it is possible to hydrolyse coffeegalactomannans using an immobilized mannanase extracted from Aspergillusniger.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to an isolatedβ-mannanase protein having an amino acid sequence which is 90% similarto the amino acid sequence of SEQ ID NO: 1. The present invention alsorelates to an isolated polynucleotide encoding the β-mannanase protein,and an isolated expression system and host cell containing thepolynucleotide.

Another aspect of the present invention is directed to a method ofrecombinantly producing β-mannanase protein. This method involvesproviding a host cell containing the polynucleotide of the presentinvention and culturing the host cell under conditions effective for thehost cell to express β-mannanase protein. The β-mannanase protein isrecovered.

A further aspect of the present invention is directed to a method ofdegrading mannans and polysaccharides in plant material. This methodinvolves providing plant material and contacting the plant material withthe β-mannanase protein of the present invention under conditionseffective to degrade mannans and polysaccharides in the plant material.

The present invention relates to an isolated polynucleotide sequencewhich encodes a mannanase enzyme involved in the hydrolysis of mannanpolysaccharides, including unbranched or branched mannan moleculeslinked to each other via a beta 1,4 glycosyl linkage. The polynucleotideis isolated from an insect (coffee berry borer, Hypothenemus hampei)genome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing SDS-PAGE analysis of the recombinantβ-mannanase protein according to one embodiment of the present inventionexpressed in recombinant baculovirus. Lane 1: molecular mass markers(kDa); Lane 2: Speed-vac concentrated protein; Lane 3: 6×-His taggedmannanase purified using paramagnetic particles.

FIG. 2 is a graph showing time course of AZC-galactomannan hydrolysiswith soluble recombinant β-mannanase from H. hampei. Enzymatic activitywas determined by blue color increase of absorbance across time.β-mannanase activity is mean±SE for three reactions.

FIG. 3 is a graph showing the effect of pH on the activity ofrecombinant β-mannanase from H. hampei, according to the presentinvention. The buffer was 200 mM sodium acetate pH 3.0, 4.0, 5.0, 6.0,7.0, 8.0, 9.0, 10.0, and 11.0.

FIG. 4 is a graph showing the effect of temperature on the activity ofthe recombinant β-mannanase from H. hampei. Enzyme solutions in 200 mMsodium acetate buffer pH 5.0 were incubated at various temperatures for120 minutes and the activities were measured as described in theExamples (infra).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to an isolated β-mannanaseprotein from coffee berry borer (Hypothenemus hampei) having an aminoacid sequence of SEQ ID NO: 1, as follows:

Met Thr Ala Asp Thr Leu Thr Arg Ala Leu Leu Leu Leu Leu Leu Leu 1              5                   10                  15 Arg Ala Ala AlaAla Val Pro Gly Phe Thr Val Ser Gly Thr Arg Ile             20                 25                  30 Leu Asp Ala Asn Gly Gln Glu PheMet Ile Arg Gly Val Ser His Ala         35                  40                 45 His Thr Trp Tyr Lys Asp Asp Ile Asn Gly Ala Ile ThrSer Ile Ala     50                  55                  60 Ala Ala GlyAla Asn Thr Val Arg Ile Val Leu Ser Asn Gly Gly Gln 65                 70                  75                  80 Trp Thr LysAsp Asn Leu Asp Ser Val Gln Asn Ile Leu Ser Leu Cys                 85                 90                  95 Glu Ser His Lys Leu Ile Ala MetLeu Glu Val His Asp Ala Thr Gly             100                 105                110 Asn Asp Ser Gln Glu Thr Leu Glu Asn Ala Val Asn TyrTrp Lys Glu         115                 120                 125 Leu ArgAsp Leu Leu Ile Gly Lys Glu Asp Arg Val Ile Ile Asn Ile     130                135                 140 Ala Asn Glu Trp Phe Gly Thr TrpAsp Thr Ala Gly Trp Ala Asp Gly 145                 150                155                 160 Tyr Lys Val Val Ile Pro Glu LeuArg Asn Ala Gly Leu Glu His Leu                 165                 170                175 Leu Val Val Asp Thr Ala Gly Tyr Gly Gln Tyr Pro GlnAla Ile Phe             180                 185                 190 GluLys Gly Lys Glu Val Phe Gln Thr Asp Leu Leu Ala Arg Thr Val         195                200                 205 Phe Ser Ile His Met Tyr Glu TyrAla Ala Thr Asp Val Thr Met Ile     210                 215                220 Lys Gly Asn Ile Asp Ser Ala Leu Asn Thr Gly Ile ProVal Ile Ile 225                 230                 235                240 Gly Glu Phe Gly Asp Arg Lys Pro Glu Ser Gln His ValAsp Ile Asp                 245                 250                 255Thr Ile Met Ser Tyr Thr Arg Glu Lys Ser Val Gly Trp Leu Ala Trp            260                 265                 270 Ser Trp Tyr GlyAsn Gly Asn Asp Glu Ser Ile Leu Asp Leu Thr Asn         275                280                 285 Gly Pro Ser Gly Asp Tyr Ser LeuThr Asn Val Gly Ser Gln Ile Val     290                 295                300 Asp Ser Glu Asn Gly Ile Arg Lys Thr Ser Thr Ile CysSer Ile Phe 305                 310                 315                320 Asn

The present invention is also directed to isolated β-mannanase proteinshaving an amino acid sequence which is at least 90% similar, at least91% similar, at least 92% similar, at least 93% similar, at least 94%similar, at least 95% similar, at least 96% similar, at least 97%similar, at least 98% similar, and/or at least 99% similar to the aminoacid sequence of SEQ ID NO: 1.

The polynucleotide encoding the β-mannanase protein of SEQ ID NO:1 has asequence of SEQ ID NO:2, as follows:

gctgatcggg tgtgtactca attctttaag gagtttacaa tatgaccgct gatacattaa   60cgcgggcact gctgctgttg ctgttgttgc gcgctgctgc tgctgtaccc ggattcacgg  120tttccggtac tcgaatttta gatgctaacg gtcaggaatt tatgataaga ggggtcagtc  180acgcacatac ctggtataag gatgatatta atggggccat cacatccatc gctgctgctg  240gcgccaacac ggttcgcatt gtactttcta atggcggaca gtggacaaaa gacaacctgg  300attcagttca gaacattctg tccctctgtg agagccataa gcttattgcc atgctggaag  360ttcacgatgc caccggcaat gacagccaag aaacactgga aaatgccgtg aattactgga  420aagagcttcg ggacttgctc attggtaagg aagacagagt tattatcaat atagccaatg  480agtggttcgg tacctgggat actgctggct gggccgacgg ttataaagtt gtcattccgg  540aactacgtaa cgccggactg gaacacctgc tggttgtaga cacagcggga tacggacaat  600atcctcaagc tatttttgaa aaaggtaagg aggttttcca gacagacctt cttgcccgca  660cggtgttttc cattcacatg tatgaatatg cagcgacgga tgtaacaatg ataaaaggaa  720atattgactc ggccttgaat acaggcatcc cggtgattat tggagaattt ggtgaccgaa  780aaccggagtc gcagcatgtt gatatcgata ccatcatgag ctacactcgc gagaaatccg  840taggctggtt ggcctggtcc tggtacggta acggtaacga tgaatcaatt cttgacctga  900cgaacggacc tagcggagat tacagtctta ctaacgtggg gagtcaaatt gttgacagtg  960agaacggcat tcgcaaaacc tccacaatct gttcaatatt caattaaaaa aaaagatgtt 1020tgtttgtgca tttttgttat aataaacgtt tcatttgcat att                   1063

Isolated polynucleotides having at least 90% similarity, at least 91%similarity, at least 92% similarity, at least 93% similarity, at least94% similarity, at least 95% similarity, at least 96% similarity, atleast 97% similarity, at least 98% similarity, and/or at least 99%similarity to SEQ ID NO:2 are also encompassed by the present invention.

The genomic sequence from which the isolated polynucleotide of SEQ IDNO:2 is derived has a sequence of SEQ ID NO:3, as follows:

atggttgagt tcaccaatca agaatatgca gacatgcatt tgatttatgg ccaagccaat    60ggcaattcct acgaagcgcg cagactctac gcacgtagat atcctaatcg gagactacct   120gatccaaaaa catttccaaa tattcacatt cgactatgtg aaactggaac atataaacag   180ttcagtggtt tcgaaggagt acatcaaatc gcgagaactc cagaaatcga agaagccgtt   240ctaaatagtg ttgaagccga tcctgctacg agcacaagga aaattgcaat aacattgaac   300atttcattta tgcttgtctg aagaattctg actgataacc ttttgtatcc ttaccacctt   360acaagggttc aagctcttct cccacgagac tttcctttag gcgtaaattt ttgcgagtag   420ttcttacaaa tgctggctca aaatccgtcg tttgcatcgt ttgcgtcgtt ttgtttattt   480tatttacgga tgaagcaaat ttttcaagaa attccatccg aaattttcat aatgaacatt   540tttggggaga agaaaatcca catttagtac gagaaaacaa ttttcaacat caattttctg   600tcaacgtttg ggcaggaatt attggcgatt atttaatagg accatttttt ctgtcgaaga   660ggttgaatgg tggctattat catcggtttt tcgaagagga acttcccgta cttttagatg   720aggtaccgct tcttttgaga aaccaaatgt ggctaatgca cgatggtgcg ccagtccatt   780ttagtcggga agtaagggag ttcctaaatg aacattatca caaccgttgg attgatcgag   840ggggaactca gtcatggccc ccgaggtccc cggacctgaa tagtctggat ttttttttct   900agggacatct caaatccttg gtgtaccaaa ccccaattaa cacagtggag gaattgcgaa   960acagaatagt cgattcatgt aacgtcattc gcaatactcc tggtattttt gaaagagtcc  1020gccggtctat gaggcacaga gcggaatctt gcatcttagc aagaggagga cattttcaac  1080agttcctata gtcttgtttt atttagatta aattactttt actgttacct tacgaattta  1140atacataaga ttcattgtac tcttttgttg tacgttttcc ttaatatgca tcggtaactg  1200tttatgcaaa tttttcgcaa atgataaaag atacgagaaa aatgcaagag atcaaaaagt  1260aagagaaata gacaaggaat ctaaatgtga aatcaaaatt tatacatagt gttccaaaaa  1320aaagttagga agcaaaaaat agcacatgac ccaagaaaac ataagaccct gtatatggag  1380agcaacgatt ttgccatttt catatagagg ctcttaaaaa atatagatat accaaatttc  1440attaaattat ctttaggcat aagaaagaaa atagtagaaa atttaaaaaa aatcaaactt  1500tatcaccctg tatatcaaaa atggtgcgtt tttccccata ggtgtattag catttttttc  1560ttattttgcc gaatactatc accccctgaa atatctccat gatgatctgt tacaccctgt  1620acacctaaaa agtaaaataa taaaacgttt aaattttatc ttttaacgta gataagattt  1680tgcgtccttt gtttccttct aagttttaat cgagatttcg cctcattttc gctcattcgc  1740cagaagacct cagtgaaagc gattcattaa gtctgaaatt taactttgtt ccctaccgaa  1800tattcttttt ctgacgatag acgatagctg atcgggtgtg tactcaattc tttaaggagt  1860ttacaatatg accgctgata cattaacgcg ggcactgctg ctgttgctgt tgttgcgcgc  1920tgctgctgct gtacccggat tcacggtttc cggtactcga attttagatg ctaacggtca  1980ggaatttatg ataagagggg tcagtcacgc acatacctgg tataaggatg atattaatgg  2040ggccatcaca tccatcgctg ctgctggcgc caacacggtt cgcattgtac tttctaatgg  2100cggacagtgg acaaaagaca acctggattc agttcagaac attctgtccc tctgtgagag  2160ccataagctt attgccatgc tggaagttca cgatgccacc ggcaatgaca gccaagaaac  2220actggaaaat gccgtgaatt actggaaaga gcttcgggac ttgctcattg gtaaggaaga  2280cagagttatt atcaatatag ccaatgagtg gttcggtacc tgggatactg ctggctgggc  2340cgacggttat aaagttgtca ttccggaact acgtaacgcc ggactggaac acctgctggt  2400tgtagacaca gcgggatacg gacaatatcc tcaagctatt tttgaaaaag gtaaggaggt  2460tttccagaca gaccttcttg cccgcacggt gttttccatt cacatgtatg aatatgcagc  2520gacggatgta acaatgataa aaggaaatat tgactcggcc ttgaatacag gcatcccggt  2580gattattgga gaatttggtg accgaaaacc ggagtcgcag catgttgata tcgataccat  2640catgagctac actcgcgaga aatccgtagg ctggttggcc tggtcctggt acggtaacgg  2700taacgatgaa tcaattcttg acctgacgaa cggacctagc ggagattaca gtcttactaa  2760cgtggggagt caaattgttg acagtgagaa cggcattcgc aaaacctcca caatctgttc  2820aatattcaat taaaaaaaaa gatgtttgtt tgtgcatttt tgttataata aacgtttcat  2880ttgcatatta aatatactaa tccaatatat atttatagac aatagattat taaaaaagta  2940aattttaaaa taacttcttc aaaaaagaac atttacgctc aaagtgacct atagacgtca  3000ataatttaaa atgtcactct tcgcacattg acaataacct gcatagacgt ctatgaacgt  3060cgttgtctat agacgtgttc ctttaattgt tttctaaagc tttgatcaat tggttcagaa  3120aaacggttca atagattcat ttaataattt acaggactat tgggggtaca ttaggctata  3180aaacggcctc tcaatatttg tcttcccatc aatatttaaa aagtaatagt agatttgtta  3240aaggactgta aaatgtaatt ttttagtagt ttttccaaat taaagctaag agtaaaaaaa  3300acggtttttc tacaaaagtc atggaagggt tttgtaggga atttaatcag gtttttaaaa  3360ctatccttga aattaaagtt tacttaagcg atcactggtt gctgagatat cgatgatcaa  3420agataaaagg atcctttttc tttcaaagtt agatgtctca gcaagggatt gacgtagatg  3480tatgaaaaaa aaaacaaaat gaagctgaat aaacaaggta accgaccact gtacgacaag  3540ggttcaaatg gaaaaaaatt tctgagaccc atggagactt tagaagaaga agaaaatttt  3600gaaaaatgtt tacctcgcgc catttcttgg gattgcgcgg taaccataac tccaaaggaa  3660attccgatgt aagatctgaa aactataaaa cattaagctt caaaatgctt ttttctccaa  3720ctcgatacga ccgttttttc acaaagatac tcaaagaaca ctaaaaaaaa taaaaaaagg  3780tttttacttt aattttttgg attagtatta ttaacattat ttaatctaaa ataatactga  3840tattggtatt aactttcacc aggtacactg gtttcaatag aaacgttacc aatttagtta  3900catagcatca aagaaaagaa tgacattatg atcatcaatt ataattgatt gttcgattat  3960aatataacta ttattgatta ttatattatt ataattctct taggtattaa gcccttaagt  4020caaaaatcgt agttttctac aaaacgagat taaaaatttt ataacgctat gcaacagaaa  4080aaaaaattca ctgggtttac agtacgtggt gatgatacct cactatttac tcaattaata  4140tttatatata aaatagtccc atcaattatt taaattttca aaaaaaaaat ttattaaatt  4200gttcaacaaa gagttaacaa taatttcaca atagttaaga actaatttct taattttcaa  4260tatggccccc ttcctgtaaa atacacttat ctattcgttt tctgatgtgt tgcacaactt  4320aaataataaa tgttgtaata tttcttgaaa atttaattaa ggttaccaga tgtcactttt  4380ttacaattat tctaacaaga gttgagtgat agtttagggt tcgatccctg ctacctccga  4440tatttttttt tttcgttttt tttttgttaa taacaatagt aataattgtc aaaataatta  4500aaaacgataa aaataattta tttgacgcat tttacagtta tttaaagctt gtaatgagag  4560aatttatatg attcgcatat taaattaagg attttcacta caaatttcat atttcaaaaa  4620caattggtcc tattttaata aaattatcta ccaggaggtt tttgatgatg ctctttcata  4680atatgttaaa aaaatgcgtt taaaattacc ctaatacatt tttctgtaaa atctacccta  4740atatttaact ataaaaacgt acgccaatga cgaagggaaa ccattttagg caaatcacaa  4800tcggaattca aagatacaca actgatccaa atttgaagtg aatcggctaa acagtttttg  4860agatacaaaa gtggctccat gaatcgtgcg acatactata tgcgatcaaa ataagacttt  4920tttttcctat aaacatgtgc cctaaaatgc accccctcca aactacagcc attctaagtt  4980gcgcgacaaa aatcaattat tttaaatttt gactacagtt atggatcaaa tttttgaaaa  5040attgcacatc cgtttttctt aaacactgta ttcatttctt gctttaaaaa taacgtaacc  5100ctaattttag agaagatgga gagggatcca cttattgcgt aaataaaatt taatgtttcc  5160aacgagaaat cctaacttta accatcagca tctagtagac catggaaaaa tctaaaattt  5220atctctagca ttttcttaat gcaactaatc gaatctttac aggacctgac ataaaaaaat  5280taattgatga tacctctttt ttatcaagtc tcatttacat agaataacag gcatgattag  5340catttgttga cgtcacaaaa aattttctcg gcaattacaa atcaacagat ttctgtgaaa  5400aaattaattt aatgttgaat gcctatcaga aattagggtg caatatatca ctgaaaattc  5460atttcttgca atatcactta agttttttcc gaaaaatatg gattcagtta gtaatgaact  5520aggtatatgt gcggtaaatt tatgttgcaa aaaaatgtac gcagattacc tgaattataa  5580caacaaagtt gtcgaaaaat gtcgaaacac ctccgaaaat tgaaacatag caaaaattat  5640ccgagtcatt tatgcacttg aattcgtgat ttcttttact catttcacaa atttattaca  5700tgattaaaat taaattattt attaaattac aagagaatga taaaaaaaat aattaaggct  5760tttaaatgtt gtatatgaac tgtcacaccg taacagattt gtcaacatat taacaattga  5820cagtaaaaat ttcaaattta taattcggtc atccattaga aaattcataa cttcactatt  5880tatccataaa tttgcatcca aagtaacttg ttcttttcat gaatgtgtca gctctacata  5940aaacaattga aactggacct atctttgcga tttctatttt ttccagggcc atatgggaaa  6000ctttgcaata aatcttgtgc aataaattat gacaattagg aatatttggt gcggtgtaca  6060tattatatac aggttgaaga aaatacctcc cccataaaag ggccttcaaa atagtctaat  6120acaattttgc ccttaaacga acagggaaga taatattaaa ggatattgaa atttatatgg  6180gttttcctat tgtcggtccg gaggggcaac atccaatata ctatatgaaa taaagttgtc  6240tatagttagt acattgttaa taatttgatt ttcatatatt ctgtattcag tttctaccgt  6300acaagtagcg gcggagatgt ggatgtcatg tattattaaa cttgtatgga aaaaaaggat  6360aaaaaaggac acttatatct ccaccattga tgaatggtta aagctggtaa ttttttggaa  6420tacacctacc gatatgctaa actcatatac gaaatttggt taaaaaatct taagccgttt  6480tggaatttaa aaaataaaga aatgttcttt tttttaaact ttaacaccct gtatctcgga  6540aacggtgcgt ttgcgggccc atgttcatat aaactttttt gcttattttt gtctgaagaa  6600tcatcccttg aaatttatgc acgtacttaa ttaacaccct gtatttcacg taccatgtcc  6660tactatacct aaactcaaca gttggcgaga aattttgttg tcgtacctgg tttttgggcc  6720atcctgtata catggattta tcatcacaaa aacctcaacc aaaaataaaa attgacgtgt  6780atgagcgttt tttttttttg aaaaacaaat ttctgaattt taaatgaatg tattccttat  6840tttatcataa acctgtatat aaatttaaaa aaaacgtgat gtgatacgtg aaaactaagt  6900tgttatttgg attcagcagg acaaaattta tatgttttat ctaaaattat gcaaaaaata  6960ttttcatcgc agacccgtgt tatcaatgta aaaattaatt aaaccccctc gtaaaagcca  7020gcgttttgac atttgaatgt ttccgcccca atgttgaagg gaaaaataaa aagttactag  7080aatgtaacta gttaggctgc catatttgga gtaacatgtt ccctctctct ctctaacaca  7140cgtgaacata actttgcggt actgtataga tttagtgact gtacctacat agtcatacgt  7200atggaaactt atacagtgta tcaatttaaa aactagcaat ggagaatatc ttctaaatga  7260aaagtgctat caggacgatc tcaaaaacgt atcgggggat tcgaaaagga accaaaatga  7320tatattaatc aatccgtttt cacttatccc ctcgccccca accaccccaa cgttcagaat  7380ttcaaatggc accatctgtc atgtaatacc tcaaatggaa ggtatttcaa aactgcattt  7440aggggtataa ttagatttta atttattgat tcgtttttga gaaaaagtgc ctcaaaaagg  7500taaaattaaa aattttaata ttgtttctta caaaaacaat taggttttca atacatgttg  7560ttaggaacag ggccggattt agggcagggc aggcggggct actgccccgg ggcctccaca  7620aggaaggggc ccccacaata gagaaaaata aatatcgaaa tttcgacggg tcagcaactt  7680ttaatttttt ttagtttttt caatacttaa gtgtctttcg ggacccgtgg agcgaattgg  7740ggtgctgggt ggggtaaaca actagctccg agtgtgctaa ttgcggcgtt ctttaaaaaa  7800aataatttat ttttgtttct ttttttcatt ttctggcgtt tcctggatgc caaaaatatg  7860ttttttttct caaaactgta aatgtcgtta aagcgttacg gaccccaagt ttccatttac  7920tgtcaatttt ttattttatc aagcatctcg gtggcaatac ttttttttta atgtgtaatt  7980tgtaatttgt gctcatgagt taaaaagaaa taaaataagt aaaaagaaat tccaaggttt  8040tataaaaagt atatatggta accgggtgac tattaaaatt tcttctcaag taggctttgg  8100aattatttga caccgagtcc gtatgtctta gatttattaa cagattcgta tttaagtgag  8160gttgggttta tttgtttata ttcgaaacta agtaaagaac atatcaaaga cttacggact  8220cgctgtcaaa taattccaca gcctacttga gaagaaattt taatagtcac ccgttataaa  8280tacgatttaa atcttacggt tggcaacact gtcgtggtta agaagtgtcc gagtgtagcg  8340tgctcgaagc ggggaatccc taaattttat tagaagctac ggacgtattg caacaaaagt  8400aaaatcttct gaatttatat ctacaaacac acgtaagtat tttcgtattt atcttgtgat  8460ctataagata taatgaattt tatatttcat tgtgcaatta cgaaaatttg tttttttttc  8520ttaaaggagc gatatgcttt aaagagcaaa aacgtaaaca gaggttatgg aggttttatt  8580ccacgactac attcttattt tcttaagatt tcttttacaa tggtattatg agtgatcgcc  8640ataaaactcc tcgagttttc gctagtggat cccaaaaaag gaaattgcaa acagaacgtg  8700aaaaaaaaaa agtgaagaaa atttagctaa aatacccaaa ttgaccaact attttacatc  8760gacacccaaa caaaaacttc cgcaagatcc tgaaaaatca gcagaagatt cagcagtaga  8820tggagatggg gttgatagta atcaagataa tccagctgtt acatcaggcg acaccatagg  8880atcttcaaaa acttgtagtc acaatcaaga ggaagtagat tttcgtggtt tcaaaaatga  8940cattggtctt tggcctgacg tcataacaga agaaatgatc aaatattggg cgaagaaggg  9000ttccacaaaa ctgcaaaact gtgatgaagt ttctctgcag aattcagttc tccaagacca  9060gtcgcaagat aataaaaact ttgttcggaa atgttcaaag aatatgttta cacgtcgcaa  9120tcaaaatcaa gagactgtta atcgattctg gctttgtttt tctccaacta agggaaaagt  9180atattgctat gcatgtaaat taatgtccac tcaaaaacga agctaagtgg ggaaggcttc  9240agtgactgga aacatgcatc tgagcggctg tacgagcatg agatttcaaa aactcatttg  9300gaatcagtga tgaatttagt gcaacgagga gaagtcacag gacgtatcga tcaagagtta  9360acgatacaag aggcacaaca aattgaatat tggcgaaaaa ttcttacaat tgtcgtcagt  9420acgattaaat tcattgctga acgcggatta gcctttcgag gagacgatga aattattgga  9480tcatcgagaa atggcaattt tctgggtatt ttagaattgc tagccgagta cgaaccaatc  9540ttggcagctc atttaaaaca gcatgcaaac aaagggagag gtcacgtcaa ttatctttct  9600tctacgatct gcgaagaact gacaaattcc atgggtgatc aagtgttcaa tgaaatcgta  9660gcaaggatta aaaaatcaaa gtactattct gtttcagtgg actctactcc tgacgaatct  9720catatcgatc aacttactat agttattcgc tatattgaag gatcgatgcc aaaggaacga  9780tttcttattt tttaccaaat tgcggtcata ctggtgaagc cacagcaaaa gctttactac  9840aatttttaag ttaccatcaa attgacatcc ttaattgccg aggtcaatcg tacgacaatg  9900ctgcaaatat gagtggtaaa tatcaaggga tgcaagctct tattttgcag aaaaatcatt  9960tatctacgtt tgtaccatgt tgtggtcact cactcaactt agttggaaag gcagctgcta 10020actcttgtgc atcggcagtt caattctttg atttcgttca gaatttatat acgtttttta 10080cagcaagtac acaacgatac cgaattctgt ctgaaaaatt atcagagaaa aaaagcggac 10140agtcatatgt tttaaaaaat cttagcgata ctcgctggtc atgtagggtt gcagccacga 10200aggccattgt tatgggatat tctgaaatcg aagaagctct aaccagcata tcttctgata 10260aggaacagaa agat                                                   10274

The determination of percent identity, i.e. sequence similarity, betweentwo amino acid sequences or two nucleotide sequences can be accomplishedusing a mathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin et al., “Methods for Assessing the StatisticalSignificance of Molecular Sequence Features by Using General ScoringSchemes,” Proc. Natl. Acad. Sci. 87:2264-2268 (1990), which is herebyincorporated by reference in its entirety, modified as in Karlin et al.,“Applications and Statistics for Multiple High-Scoring Segments inMolecular Sequences,” Proc. Natl. Acad. Sci. 90:5873-5877 (1993), whichis hereby incorporated by reference in its entirety. Anothernon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers et al., CABIOS (1989).Such an algorithm can be incorporated into the ALIGN program (version2.0) which is part of the CGC sequence alignment software package.Additional algorithms for sequence analysis are known in the art andinclude ADVANCE and ADAM as described in Torellis et al. “ADVANCE andADAM: Two Algorithms for the Analysis of Global Similarity betweenHomologous Informational Sequences,” Comput. Appl. Biosci. 10:3-5(1994), which is hereby incorporated by reference in its entirety, andFASTA described in Pearson et al., “Improved Tools for BiologicalSequence Comparison,” Proc. Natl. Acad. Sci. 85:2444-8 (1988), which ishereby incorporated by reference in its entirety.

The isolated β-mannanase protein of the present invention is preferablyproduced in purified form by conventional techniques. For example, toisolate the protein, a protocol involving a host cell such as Escherchiacoli may be used, in which the E. coli host cell carrying a recombinantplasmid is propagated, homogenized, and the homogenate is centrifuged toremove bacterial debris. The supernatant is then subjected to sequentialammonium sulfate precipitation. The fraction containing the β-mannanaseprotein can be subjected to gel filtration in an appropriately sizeddextran or polyacrylamide column to separate the protein or polypeptide.If necessary, the protein fraction may be further purified by HPLC.Isolated β-mannanase proteins of the present invention may also beproduced according to a protocol involving insect host cells, preferablySf9 insect cell lines.

The present invention is also directed to fragments of the β-mannanaseprotein of the present invention. Fragments of the β-mannanase proteincan be produced by digestion of a full-length protein with proteolyticenzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin.Different proteolytic enzymes are likely to cleave the β-mannanaseprotein at different sites based on the amino acid sequence of theprotein.

In another approach, based on knowledge of the primary structure of theprotein, fragments of the genes encoding the protein may be synthesizedby using a PCR technique together with specific sets of primers chosento represent particular portions of the protein of interest. These thenwould be cloned into an appropriate vector for expression of a truncatedpeptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for theprotein being produced. Alternatively, subjecting a full lengthβ-mannanase protein of the present invention to high temperatures andpressures will produce fragments. These fragments can then be separatedby conventional procedures (e.g., chromatography, SDS-PAGE).

Variants may also (or alternatively) be made, for example, by thedeletion or addition of amino acids that have minimal influence on theproperties, secondary structure and hydropathic nature of the protein.For example, a protein may be conjugated to a signal (or leader)sequence at the N-terminal end of the protein which co-translationallyor post-translationally directs transfer of the protein. The protein mayalso be conjugated to a linker or other sequence for ease of synthesis,purification, or identification of the protein.

The protein of the present invention is preferably produced in purifiedform (preferably at least about 80%, more preferably 90%, pure) byconventional techniques. Typically, the protein of the present inventionis secreted into the growth medium of Helicobacter cells or host cellswhich express a functional type III secretion system capable ofsecreting the protein of the present invention. Alternatively, theprotein of the present invention is produced but not secreted intogrowth medium of recombinant host cells (e.g., Escherichia coli). Insuch cases, to isolate the protein, the host cell (e.g., E. coli)carrying a recombinant plasmid may be propagated, lysed by sonication,heat, differential pressure, or chemical treatment, and the homogenateis centrifuged to remove bacterial debris. The supernatant is thensubjected to sequential ammonium sulfate precipitation. The fractioncontaining the polypeptide or protein of the present invention issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the proteins. If necessary, theprotein fraction may be further purified by HPLC.

The present invention also relates to an isolated polynucleotideencoding the β-mannanase protein, and an isolated expression system andhost cell containing the polynucleotide.

Another aspect of the present invention is directed to a method ofrecombinantly producing β-mannanase protein. This method involvesproviding a host cell containing the polynucleotide of the presentinvention and culturing the host cell under conditions effective for thehost cell to express β-mannanase protein. The β-mannanase protein isrecovered.

The polynucleotide of the present invention may be inserted into any ofthe many available expression vectors using reagents that are well knownin the art. In preparing a DNA vector for expression, the DNA sequencemay normally be inserted or substituted into a bacterial plasmid. Anyconvenient plasmid may be employed, which will be characterized byhaving a bacterial replication system, a marker which allows forselection in a bacterium, and generally one or more unique, convenientlylocated restriction sites. Numerous plasmids, referred to astransformation vectors, are available for plant transformation. Theselection of a vector will depend on the preferred transformationtechnique and target species for transformation. A variety of vectorsare available for stable transformation using Agrobacterium tumefaciens,a soilborne bacterium that causes crown gall. Crown gall ischaracterized by tumors or galls that develop on the lower stem and mainroots of the infected plant. These tumors are due to the transfer andincorporation of part of the bacterium plasmid DNA into the plantchromosomal DNA. This transfer DNA (T-DNA) is expressed along with thenormal genes of the plant cell. The plasmid DNA, pTi, or Ti-DNA, for“tumor inducing plasmid,” contains the vir genes necessary for movementof the T-DNA into the plant. The T-DNA carries genes that encodeproteins involved in the biosynthesis of plant regulatory factors, andbacterial nutrients (opines). The T-DNA is delimited by two 25 bpimperfect direct repeat sequences called the “border sequences.” Byremoving the oncogene and opine genes, and replacing them with a gene ofinterest, it is possible to transfer foreign DNA into the plant withoutthe formation of tumors or the multiplication of Agrobacteriumtumefaciens (Fraley et al., “Expression of Bacterial Genes in PlantCells,” Proc. Nat'l Acad. Sci. 80:4803-4807 (1983), which is herebyincorporated by reference in its entirety).

Further improvement of this technique led to the development of thebinary vector system (Bevan, “Binary Agrobacterium Vectors for PlantTransformation,” Nucleic Acids Res. 12:8711-8721 (1984), which is herebyincorporated by reference in its entirety). In this system, all theT-DNA sequences (including the borders) are removed from the pTi, and asecond vector containing T-DNA is introduced into Agrobacteriumtumefaciens. This second vector has the advantage of being replicable inE. coli as well as A. tumefaciens, and contains a multiclonal site thatfacilitates the cloning of a transgene. An example of a commonly usedvector is pBin19 (Frisch et al., “Complete Sequence of the Binary VectorBin19,” Plant Molec. Biol. 27:405-409 (1995), which is herebyincorporated by reference in its entirety). Any appropriate vectors nowknown or later described for genetic transformation are suitable for usewith the present invention.

Suitable vectors for practicing the present invention may also include,but are not limited to, the following viral vectors such as lambdavector system gt11, gtWES.tB, Charon 4, and plasmid vectors such aspBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339,pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/− (see“Stratagene Cloning Systems” Catalog (1993), which is herebyincorporated by reference in its entirety), pQE, pIH821, pGEX, pETseries (Studier et al, “Use of T7 RNA Polymerase to Direct Expression ofCloned Genes,” Methods in Enzymology 185:60-89 (1990), which is herebyincorporated by reference in its entirety), and any derivatives thereof.

U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is herebyincorporated by reference in its entirety, describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase. These recombinant plasmidsare then introduced by means of transformation and replicated inunicellular cultures including prokaryotic organisms and eukaryoticcells grown in tissue culture.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s) of the present invention. Primarily, thevector system must be compatible with the host cell used. Host-vectorsystems include but are not limited to the following: bacteriatransformed with bacteriophage DNA, plasmid DNA, or cosmid DNA;microorganisms such as yeast containing yeast vectors; mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus); and plantcells infected by bacteria. The expression elements of these vectorsvary in their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

The protein according to the present invention can be incorporated intoan appropriate vector in the sense direction, such that the open readingframe is properly oriented for the expression of the encoded proteinunder control of a promoter of choice. This involves the inclusion ofthe appropriate regulatory elements into the DNA-vector construct. Theseinclude non-translated regions of the vector, useful promoters, and 5′and 3′ untranslated regions which interact with host cellular proteinsto carry out transcription and translation. Such elements may vary intheir strength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used.

A constitutive promoter is a promoter that directs expression of a genethroughout the development and life of an organism. Examples of someconstitutive promoters that are widely used for inducing expression oftransgenes include the nopaline synthase (NOS) gene promoter, fromAgrobacterium tumefaciens (U.S. Pat. No. 5,034,322 issued to Rogers etal., which is hereby incorporated by reference in its entirety), thecauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Pat. No.5,352,605 issued to Fraley et al., which is hereby incorporated byreference in its entirety), those derived from any of the several actingenes, which are known to be expressed in most cells types (U.S. Pat.No. 6,002,068 issued to Privalle et al., which is hereby incorporated byreference in its entirety), and the ubiquitin promoter, which is a geneproduct known to accumulate in many cell types.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed. The inducer can be achemical agent, such as a metabolite, growth regulator, herbicide, orphenolic compound, or a physiological stress directly imposed upon theplant such as cold, heat, salt, toxins, or through the action of apathogen or disease agent such as a virus or fungus. A plant cellcontaining an inducible promoter may be exposed to an inducer byexternally applying the inducer to the cell or plant such as byspraying, watering, heating, or by exposure to the operative pathogen.An example of an appropriate inducible promoter for use in the presentinvention is a glucocorticoid-inducible promoter (Schena et al., “ASteroid-Inducible Gene Expression System for Plant Cells,” Proc. Natl.Acad. Sci. 88:10421-5 (1991), which is hereby incorporated by referencein its entirety). Expression of the transgene-encoded protein is inducedin the transformed plants when the transgenic plants are brought intocontact with nanomolar concentrations of a glucocorticoid, or by contactwith dexamethasone, a glucocorticoid analog (Schena et al., “ASteroid-Inducible Gene Expression System for Plant Cells,” Proc. Natl.Acad. Sci. USA 88:10421-5 (1991); Aoyama et al., “AGlucocorticoid-Mediated Transcriptional Induction System in TransgenicPlants,” Plant J. 11: 605-612 (1997), and McNellis et al.,“Glucocorticoid-Inducible Expression of a Bacterial Avirulence Gene inTransgenic Arabidopsis Induces Hypersensitive Cell Death,” Plant J.14(2):247-57 (1998), which are hereby incorporated by reference in theirentirety). In addition, inducible promoters include promoters thatfunction in a tissue specific manner to regulate the gene of interestwithin selected tissues of the plant. Examples of such tissue specificor developmentally regulated promoters include seed, flower, fruit, orroot specific promoters as are well known in the field (U.S. Pat. No.5,750,385 issued to Shewmaker et al., which is hereby incorporated byreference in its entirety). In the preferred embodiment of the presentinvention, a heterologous promoter is linked to the nucleic acid of theconstruct, where “heterologous promoter” is defined as a promoter towhich the nucleic acid of the construct is not linked in nature.

The expression system of the present invention can also include anoperable 3′ regulatory region, selected from among those which arecapable of providing correct transcription termination andpolyadenylation of mRNA for expression in the host cell of choice,operably linked to a DNA molecule which encodes for a protein of choice.

The vector of choice, promoter, and an appropriate 3′ regulatory regioncan be ligated together to produce the DNA construct of the presentinvention using well known molecular cloning techniques as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. CurrentProtocols in Molecular Biology, New York, N.Y: John Wiley & Sons,(1989), which are hereby incorporated by reference in their entirety.

The efficiency of expression can be enhanced by the inclusion ofappropriate transcription or translation enhancer elements (e.g.,elements disclosed in Bittner et al., Methods in Enzymol. 153:516(1987), which is hereby incorporated by reference in its entirety).Additionally, the gene sequence can be modified for optimal codon usagein the appropriate expression system or, alternatively, the expressionhost can be modified to express specific tRNA molecules to facilitateexpression of the desired gene.

In addition, the recombinant expression vector can contain additionalnucleotide sequences. For example, the recombinant expression vector mayencode a selectable marker gene to identify host cells that haveincorporated the vector. Moreover, to facilitate secretion of theprotein from a host cell, the recombinant expression vector can encode asignal sequence linked to the amino-terminus of the protein, such thatupon expression, the protein is synthesized with the signal sequencefused to its amino terminus. This signal sequence directs the proteininto the secretory pathway of the cell and is then usually cleaved,allowing for release of the protein without the signal sequence from thehost cell. Use of a signal sequence to facilitate secretion of proteinsor peptides from mammalian host cells is well known in the art.

Once an expression system containing a polynucleotide according to thepresent invention has been prepared, it is ready to be incorporated intoa host cell. Basically, this method can be carried out by transforming ahost cell with the expression system of the present invention underconditions effective to yield transcription of the DNA molecule in thehost cell. Recombinant molecules can be introduced into cells viatransformation, particularly transduction, conjugation, mobilization, orelectroporation. The DNA sequences are cloned into the host cell usingstandard cloning procedures known in the art, as described by Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSprings Laboratory, Cold Springs Harbor, N.Y. (1989), which is herebyincorporated by reference in its entirety. Suitable host cells include,but are not limited to, bacteria, virus, yeast, mammalian cells, insect,plant, and the like. Methods of transformation may result in transientor stable expression of the nucleic acid under control of the promoter.In one embodiment, a nucleic acid construct of the present invention isstably inserted into the genome of the recombinant plant cell as aresult of the transformation, although transient expression can serve animportant purpose, particularly when the plant under investigation isslow-growing.

Plant tissue suitable for transformation include leaf tissue, roottissue, meristems, zygotic and somatic embryos, callus, protoplasts,tassels, pollen, embryos, anthers, and the like. The means oftransformation chosen is that most suited to the tissue to betransformed.

Transient expression in plant tissue is often achieved by particlebombardment (Klein et al., “High-Velocity Microprojectiles forDelivering Nucleic Acids Into Living Cells,” Nature 327:70-73 (1987),which is hereby incorporated by reference in its entirety). In thismethod, tungsten or gold microparticles (1 to 2 μm in diameter) arecoated with the DNA of interest and then bombarded at the tissue usinghigh pressure gas. In this way, it is possible to deliver foreign DNAinto the nucleus and obtain a temporal expression of the gene under thecurrent conditions of the tissue. Biologically active particles (e.g.,dried bacterial cells containing the vector and heterologous DNA) canalso be propelled into plant cells. Other variations of particlebombardment, now known or hereafter developed, can also be used. Anappropriate method of stably introducing the nucleic acid construct intoplant cells is to infect a plant cell with Agrobacterium tumefaciens orAgrobacterium rhizogenes previously transformed with the nucleic acidconstruct. As described above, the Ti (or RI) plasmid of Agrobacteriumenables the highly successful transfer of a foreign nucleic acidmolecule into plant cells. Another approach to transforming plant cellswith a gene which imparts resistance to pathogens is particlebombardment (also known as biolistic transformation) of the host cell,as disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, allto Sanford et al., and in Emerschad et al., “Somatic Embryogenesis andPlant Development from Immature Zygotic Embryos of Seedless Grapes(Vitis vinifera),” Plant Cell Reports 14:6-12 (1995), which are herebyincorporated by reference in their entirety. Yet another method ofintroduction is fusion of protoplasts with other entities, eitherminicells, cells, lysosomes, or other fusible lipid-surfaced bodies(Fraley et al., Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which ishereby incorporated by reference in its entirety). The nucleic acidmolecule may also be introduced into the plant cells by electroporation(Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985), which ishereby incorporated by reference in its entirety). In this technique,plant protoplasts are electroporated in the presence of plasmidscontaining the expression cassette. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the plasmids. Electroporated plant protoplasts reform the cell wall,divide, and regenerate. The precise method of transformation is notcritical to the practice of the present invention. Any method thatresults in efficient transformation of the host cell of choice isappropriate for practicing the present invention.

After transformation, the transformed plant cells must be regenerated.Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III(1986), and Fitch et al., “Somatic Embryogenesis and Plant Regenerationfrom Immature Zygotic Embryos of Papaya (Carica papaya L.),” Plant CellRep. 9:320 (1990), which are hereby incorporated by reference in itsentirety.

Means for regeneration varies from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining explants is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these threevariables are controlled, then regeneration is usually reproducible andrepeatable.

Preferably, transformed cells are first identified using a selectionmarker simultaneously introduced into the host cells along with thenucleic acid construct of the present invention. Suitable selectionmarkers include, without limitation, markers encoding for antibioticresistance, such as the nptII gene which confers kanamycin resistance(Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), which ishereby incorporated by reference in its entirety), and the genes whichconfer resistance to gentamycin, G418, hygromycin, streptomycin,spectinomycin, tetracycline, chloramphenicol, and the like. Cells ortissues are grown on a selection medium containing the appropriateantibiotic, whereby generally only those transformants expressing theantibiotic resistance marker continue to grow. Other types of markersare also suitable for inclusion in the expression cassette of thepresent invention. For example, a gene encoding for herbicide tolerance,such as tolerance to sulfonylurea is useful, or the dhfr gene, whichconfers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104(1983), which is hereby incorporated by reference in its entirety).Similarly, “reporter genes,” which encode for enzymes providing forproduction of an identifiable compound are suitable. The most widelyused reporter gene for gene fusion experiments has been uidA, a genefrom Escherichia coli that encodes the β-glucuronidase protein, alsoknown as GUS (Jefferson et al., “GUS Fusions: β Glucuronidase as aSensitive and Versatile Gene Fusion Marker in Higher Plants,” EMBO J.6:3901-3907 (1987), which is hereby incorporated by reference in itsentirety). Similarly, enzymes providing for production of a compoundidentifiable by luminescence, such as luciferase, are useful. Theselection marker employed will depend on the target species; for certaintarget species, different antibiotics, herbicide, or biosynthesisselection markers are preferred.

Plant cells and tissues selected by means of an inhibitory agent orother selection marker are then tested for the acquisition of the viralgene by Southern blot hybridization analysis, using a probe specific tothe viral genes contained in the given cassette used for transformation(Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Cold SpringHarbor, N.Y.: Cold Spring Harbor Press (1989), which is herebyincorporated by reference in its entirety).

After the fusion gene containing a nucleic acid construct of the presentinvention is stably incorporated in transgenic plants, the transgene canbe transferred to other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed. Once transgenic plants of this type are produced, the plantsthemselves can be cultivated in accordance with conventional procedureso that the nucleic acid construct is present in the resulting plants.Alternatively, transgenic seeds are recovered from the transgenicplants. These seeds can then be planted in the soil and cultivated usingconventional procedures to produce transgenic plants.

The present invention can be utilized in conjunction with a wide varietyof plants or their seeds. Suitable plants include dicots and monocots.Useful crop plants can include: alfalfa, rice, wheat, barley, rye,cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea,chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, tobacco, tomato, sorghum, papaya, sugarcane, and coffee.

A further aspect of the present invention is directed to a method ofdegrading mannans and polysaccharides in plant material. This methodinvolves providing plant material and contacting the plant material withthe β-mannanase protein of the present invention under conditionseffective to degrade mannans and polysaccharides in the plant material.

In a preferred embodiment, the plant material which is contacted withthe β-mannanase protein of the present invention is coffee beans,although other plant materials where degradation of mannans andpolysaccharides is desired may also be contacted.

It may also be desirable, pursuant to this method of the presentinvention, to recover soluble sugars from the degraded plant material,particularly those that result from the step of contacting the plantmatter with the β-mannanase protein.

These aspects of the present invention are further illustrated by theexamples below.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention, but they are by no means intended to limit its scope.

Example 1 β-Mannanase Cloning from Coffee Berry Borer (Hypothenemushampei) Midgut

Sample Preparation

Coffee seeds were infected with adult insects of Hypothenemus hampei(Coleoptera: Scolytidae) as described in Rubio et al., “Morfologia DelSistema Digestivo de Hypothenemus hampei (Ferrari),” Cenicafe (Columbia)58:66-74 (2007), which is hereby incorporated by reference in itsentirety. Briefly, infected coffee beans were dissected using astereomicroscope and the larvae were collected in a Petri dish. Eachlarva was stored at 4° C. for at least 10 minutes to decrease physicalactivity and then placed in a glass slide with a drop of steriledistilled water and dissected under a Zeiss Estemi 2000 stereomicroscopeusing small forceps and 0.15 mm teasing needles. In order to isolate themidgut tissue, each larva was dissected with an incision at theprothorax and the mesothorax level, then a large incision along thelarvae length to expose all the alimentary canal. Finally, after theremoval of fat tissue, the midgut region was dissected and immediatelydeposited into a pre-chilled microcentrifuge tube with 100 μl of steriledistilled water containing 0.1% RNA Later™ reagent. All the dissectedmidgut tissues were stored at −80° C. until protein and RNA extraction.

Coffee Berry Borer YSST Library Construction

RNA was extracted from the midgut tissues and stored at −80° C.Polyadenylated RNA was isolated from the total RNA using Oligotex mRNAMidi Kit(Qiagen, Valencia, Calif.) according to the manufacturer'sinstructions. The first-strand cDNA were synthesized from mRNA usingN6-NotI primer:

5′-GAGAGAGAGAGAGAGAACCGCGCGGCCGCCNNNNNN-3′ (SEQ ID NO:4)

with a cDNA Synthesis Kit (Stratagene). After second-strand synthesis,the cDNAs were prepared for unidirectional cloning by ligation withEcoRI adapters according to the manufacturer's instructions, followed byNotI digestion. The cDNAs were then fractioned on a 1% agarose gel byelectrophoresis and those within an estimated size range of 300-1,000 bpexcised from the gel and purified using QIAquick gel extraction kit(Qiagen, Valencia, Calif.). The fragments were ligated to an equipartitemixture of the three vectors, PYSST0, pYSST 1, and pYSST2, digested withEcoRI and NotI. Electrocompetent TOP10F′ Escherichia coli cells(Invitro, Carsbad, Calif.) were transformed with approximately 1 μg ofthe resulting YSST library by electroporation (MicropulserElectroporator, Bio-Rad, Hercules, Calif.) and spread on 10-15 large LBplates. Plasmid DNA was isolated from a pooled sample of the resultingtransformants using the Perfectprep Plasmid Midi kit (Eppendorf). Fiftymicrograms of the YSST library was transformed into the yeast(Saccharomyces cerevisae) strain DBYα2445 (MATα, suc2Δ-9, lys2-801,ura3-52, ade2-101) using the YEASTMAKER Yeast Transformation System2 (BDBiosciences, San Jose, Calif.). Transformants were spread on YP sucroseplates (1% yeast extract, 2% peptone, 2% sucrose, 2% agar, pH 6.5),incubated at 30° C. for 4-9d, and visible colonies were re-streaked onsucrose plate followed by incubation at 30° C. for 2-3d. Plasmids wereisolated from visible colonies as described in Hoffmann and Winston, “ATen-Minute DNA Preparation from Yeast Efficiently Releases AutonomousPlasmids for Transformation of Escherichia coli,” Gene 57:267-272(1987), which is hereby incorporated by reference in its entirety,transformed into XL 1-blue electrocompetent E. coli, and purified usinga Qiaprep kit (Qiagen). Plasmid inserts were sequenced using a primercorresponding to ADH1 promoter of pYSST0, pYSST1, pYSST2(5′-TCCTCGTCATTGTTCTCGTTCC-3′) (SEQ ID NO:5) at the Bio Resource Center,Cornell University, Ithaca, N.Y. (World Wide Web.brc.cornell.edu).

RACE Amplification

An oligonucleotide primer derived from the DNA sequence corresponding tothe predicted signal sequences of isolated mannanase YSST clone was usedin 3′RACE with the primer as an adapter primer. To obtain thefull-length cDNA sequence, ‘touchdown’ PCR was performed using a programwith 35 cycles of 94° C. for 1 min, 63° C. for 1 min, 72° C. for 2.5 minwith the annealing temperature decreasing by 1° C. every second cycle to60° C., followed by final extension of 72° C. for 10 min. The PCRproducts were subcloned into the pGEM-T Easy vector (Promega, Madison,Wis.). DNA sequences were determined as describes supra. β-mannanasefrom H. hampei is an endoglycanase (endo-β-1,4-D-glucanase, EC 3.2.1.4).It is a single polypeptide chain of 320 aminoacids, with a predictedmolecular mass of 35.62 kDa and the theoretical pI is 4.72, calculatedfrom amino-acid composition.

Example 2 Heterologous Expression of β-mannanase in Spodopterafrugiperta (Sf9) Cells

Construction of Fusion Protein Expression Plasmid

For the construction of C-terminal Mannanase-HIS-tagged expressionplasmid, the cDNAs were reamplified by PCR using a mannanase cDNA inpGEM-T Easy vector (Promega, Madison, Wis.) as a template with thefollowing primers: 5′-CACCATGGAACCTTTTGTGGTC-3′ (SEQ ID NO:6) and5′-GACAGGGATGAAGCAGATCTGG-3′ (SEQ ID NO:7). The underlined portion ofSEQ ID NO:6 was introduced for directional TOPO cloning. The latterreverse primer lacks the stop codon in the native β-mannanase cDNA. Theresulting cDNA was cloned into pENTR/β-TOPO vector (Invitrogen,Carlsbad, Calif.) and designated pENTR/β-Mann.

DNA Baculovirus Recombination and Transfection

Baculovirus construction and protein expression in Sf9 cells wereperformed according to the BaculoDirect Baculovirus Expression Systemprotocol from Invitrogen (Carlsbad, Calif.). Spodoptera frugiperda Sf9cells were transfected with recombinant bacmid DNA for production of thebaculovirus particles. Cells were cultured at 27° C. in SF900-II medium(Life Technologies) supplemented with 100 U/ml penicillin and 100 μg/mlstreptomycin. For transfection, 9×10⁵ cells were plated in 35-mm tissueculture flasks and incubated for 1 h in 2 ml Sf900-II SFM (LifeTechnologies) without antibiotics to allow adhesion of the cells to thedish. The medium was then changed to 1 ml serum-free Sf900-II withoutantibiotics, containing recombinant bacmid DNA (5 μl of a standardmini-preparation of plasmid DNA) that had been pre-incubated for 30 minat room temperature with CellFectin (6 μl) (Life Technologies). Cellswere incubated with the liposome-DNA complex at 27° C. for 5 h. Thetransfection medium was removed and 2 ml of SF900-II medium, containingantibiotics, was added. PENT™/Man plasmid was transfected into Sf9 cellsand nonrecombinant bacmid (Bd) DNA and PENT™/CAT were used as,respectively, negative and positive controls. Transfected cells wereincubated at 27° C. for 72 h allowing baculovirus production and releaseinto the culture medium. The culture medium from each transfection wascollected, clarified (500 g for 5 min), and stored at 4° C. as a mastervirus stock. Transfection efficiency, recombinant baculovirus (Bv-Man)and nonrecombinant baculovirus (Bv) production were monitored byvisualization of the cytopathic effect displayed by transfected cellswithin 48 h after subculturing under a phase contrast microscope andassaying the presence of baculovirus DNA through PCR analysis. To thisend, baculovirus present in 50 μl of infected culture supernatant wassedimented at 12 000 g for 10 min in a microcentrifuge tube, and avolume (25 μl) of proteinase K buffer (10 mM Tris-HCl, pH 7.8; 5 mMEDTA; 0.5% SDS) containing 50 μg/ml of proteinase K (Sambrook et al.,1989) was added to the pellet to digest viral proteins for 1 h at 56° C.An additional heating at 95° C. for 20 min was included in order toinactivate the enzyme before proceeding to the PCR step. Viral DNAamplification was carried out using 2 μl of this DNA preparation as thetemplate at the same conditions and primers described above. The cellswere selected with ganciclovir for 120 hours, and the resulting viralstock was amplified twice by infecting the Sf9 cells.

Amplification of Baculovirus Stocks

For amplification of the baculovirus master stocks, 1×10⁶ Sf9 cells wereplated in a 25-cm² flask and incubated for 1 h with 10 μl of baculovirusmaster stock in 1 ml of SF900-II medium containing antibiotics(corresponding to an MOI of 0.01-0.1). After this period, the medium wascompleted to 4.5 ml and the infected cells were incubated for 48 h at27° C. The culture medium was collected, clarified (500 g for 5 min),and stored at 4° C. as viral stocks for recombinant protein production.

Scale Up Production of Recombinant β-Mannanase

Cells of S. frugiperta were plated in a 225 cm² with 50 ml of culturemedium and incubated as above. Cells were transfected at log phase using500 μL of a viral stock P3 (titer 3.44×10⁶ pfu/ml). After 96 hours, theculture medium was collected, clarified (500 g for 5 min), and therecombinant β-mannanase was purified using the MagneHis System®(Promega, Madison, Wis.). This resulted in the production of 2.4 mg ofpurified β-mannanase per 100 ml of culture.

Example 3 Purification of Recombinant β-Mannanase

MagneHis Ni-Particles® (Promega, Madison, Wis.) pull-down assays wereperformed according to the manufacturer's protocol. Briefly, 10 ml ofculture medium after removing cells was mixed with 300 μl of MagneHisNi-Particles® and incubated at room temperature for 2 min with gentleshaking. After incubation, the tube was placed in a magnetic stand for30 seconds to allow the MagneHis Ni-Particles® to be captured by themagnet, and the supernatant was removed. The MagneHis Ni-Particles® werewashed three times with the binding/wash buffer. Pureβ-mannanase-6×His-tagged protein was subjected to SDS/PAGE (FIG. 1) andfunctional assay (FIG. 2).

Example 4 Determination of Enzyme Activity Using β-Galactomannans fromCarob Tree Seeds

Azurine-crosslinked-Galactomannan was prepared by dyeing andcrosslinking galactomannan polysaccharide extracted from carob seedflour (AZCL-galactomannan®; Megazyme International Ireland Ltd.). TheCarob tree, Ceratonia siliqua, is an evergreen shrub or tree, native tothe Mediterranean region, cultivated for its edible seed pods. Thissubstrate is insoluble in buffered solutions, but rapidly hydrates toform gel particles which are readily and rapidly hydrolysed by specificendo-hydrolases releasing soluble dye-labeled fragments according toMarraccini et al., “Molecular and Biochemical Characterization ofENDO-β-MANNANASEs from Germinating Coffee (Coffea arabica) Grains,”Planta 213:296-308 (2001), which is hereby incorporated by reference inits entirety. An aliquot of 20 μL of the recombinantβ-mannanase-6×His-tagged protein mixed in substrate solution [1% (w/v)AZCL-galactomannan® in 0.2 M acetate buffer (pH 5.0)] was incubated at37° C. with gentle shaking. Aliquots of 200 μL were removed every 30 minand heated at 100° C. for 5 min to stop the reaction. Each aliquot wascentrifuged at 13k rpm for 5 min and the absorbance was measured at λ₅₉₅nm (FIG. 2).

Example 5 Determination of Enzyme Activity Using β-Galactomannan fromCoffee Beans

Coffee β-Galactomannan Purification

A sequential fractionation procedure based on a delignificationtreatment, an acid wash, and subsequent alkali extraction (as inBradbury et al., “Chemical Structures of Green Coffee BeanPolysaccharides,” J. Agric. Food Chem. 38:389-392 (1990), which ishereby incorporated by reference in its entirety) was used to isolatepure β-mannan from green coffee beans. Ground green Coffea arabicacoffee beans were Soxhlet-extracted with chloroform/methanol (2:1) andpetroleum ether (5h) to remove lipids and with aqueous ethanol (95%,overnight) to remove low molecular weight carbohydrate. Defatted beanswere hot water extracted and then delignified with weakly acidic sodiumchloride solution, according to the method of Wolfrom and Patin,“Carbohydrates of the Coffee Bean. IV. An Arabinogalactan,” J. Org.Chem. 30:4060-4063 (1965), which is hereby incorporated by reference inits entirety, to give a white holocellulose product. Most of thearabinogalactan polymer was solubilized, in a partially hydrolyzed form,by washing with dilute hydrochloric acid (1%, 80° C.). The mannan wasthen isolated in discrete fractions by extraction (overnight, 4° C.)with 2.5 and 10% sodium hydroxide solutions. Addition of ethanol to the2.5% NaOH extracts led to a precipitate containing arabinogalactan andmannan. Neutralization of the 10% NaOH extracts led to rapid formationof a white precipitate, which was removed by filtration after themixture was allowed to stand overnight at 4° C. A further fraction wasobtained by addition of ethanol to the filtrate. The precipitates wereall washed with ethanol and diethyl ether before drying. The mannansubstrate used in this work was the fraction precipitated byneutralization of the 10% NaOH extracts, which contained 94% mannan byweight.

Effect of pH and Temperature on the Activity of the Recombinantβ-Mannanase from H. hampei

In order to test enzyme activity against coffee galactomannans, theβ-mannanase activity was determined by the dinitrosalicylic acid assayof Bernfeld, P. In: Collowick S. P. and Kaplan N. O. (eds.), Methods inEnzymology, Vol. I, Academic Press, New York, pp. 149-158 (1955), whichis hereby incorporated by reference in its entirety. The optimal pH ofthe enzyme activity against coffee galactomannan was determined atdifferent pH values ranging from 4.0 to 11. The buffer was 200 mMsodium-acetate and 100 mM Sodium chloride at 37° C. The highest enzymeactivity was observed at pH 6.0 (FIG. 3). The effect of temperature onactivity of the recombinant β-mannanase against coffee galactomannan wasexamined in the temperature range of 15° C. to 70° C. using the samebufffer at pH 6.0 (FIG. 4). The enzyme showed maximum activity around50° C. The activity of the enzyme decreased sharply with furtherincreases in temperature.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An isolated polynucleotide encoding a β-mannanase protein having anamino acid sequence which is at least 95% similar to the amino acidsequence of SEQ ID NO:
 1. 2. The isolated polynucleotide of claim 1,wherein the polynucleotide encodes a protein having the amino acidsequence of SEQ ID NO:
 1. 3. The isolated polynucleotide of claim 1,wherein the polynucleotide has the nucleotide sequence of SEQ ID NO: 2.4. An isolated expression system containing the polynucleotide ofclaim
 1. 5. An isolated host cell containing the polynucleotide ofclaim
 1. 6. The host cell of claim 5, wherein the host cell is abacterial cell.
 7. A method of recombinantly producing β-mannanaseprotein, said method comprising: providing the host cell of claim 5;culturing the host cell under conditions effective for the host cell toexpress β-mannanase protein; and recovering the β-mannanase protein. 8.The host cell of claim 5, wherein the host cell is a eukaryotic cell. 9.The host cell of claim 8, wherein the eukaryotic cell is selected fromthe group consisting of plant cells, fungal cells and insect cells.